Modified acyl-ACP desaturase

ABSTRACT

Disclosed is a methods for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.

GOVERNMENT SUPPORT

Experimental work described herein was supported by grants from theUnited States Government which may have certain rights in the invention.

RELATED APPLICATIONS

The subject patent application is a continuation of U.S. applicationSer. No. 08/853,979, filed May 9, 1997, now U.S. Pat. No. 5,888,790,which was a continuation-in-part of U.S. application Ser. No.08/689,823, filed Aug. 14, 1996, now U.S. Pat. No. 5,705,391, thecontents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Fatty acid biosynthesis in higher plants has recently attractedincreased interest because of the possible use of plant oils asrenewable sources for reduced carbon. In plants, fatty acid biosynthesisoccurs in the chloroplasts of green tissue or in the plastids ofnonphotosynthetic tissues. The primary products in most plants are acylcarrier protein (ACP) esters of the saturated palmitic and stearicacids.

Δ⁹ stearoyl-acyl carrier protein desaturase (Δ⁹ desaturase) is a plastidlocalized non-membrane bound soluble desaturase that introduces thefirst double bond into saturated fatty acids (resulting in thecorresponding mono-unsaturated fatty acids). Recently, several relatedsoluble desaturases have been identified in the seed tissues of variousplants that possess fatty acids with unusual double bond positions.Members of this class of soluble desaturases are specific for aparticular substrate chain length and introduce the double bond betweenspecific carbon atoms by counting from the carboxyl end of the fattyacid; for instance, the Δ⁹ desaturase is specific for stearoyl-ACP, andintroduces a double bond between carbon 9 and 10. Initial desaturationreactions in animals and fungi, and subsequent desaturation reactions inplants, are mediated by a distinct class of fatty acid desaturases thatare integral membrane proteins. Since most plants lack other desaturasesthat act on the 18:0 level, the ratio of saturated to unsaturated fattyacids in higher plants is mainly controlled by enzymes which catalyzethe conversion of saturated to mono-unsaturated fatty acids. Δ⁹desaturase cDNA encode precursor proteins containing an N-terminaltransit peptide for targeting to the plastid. For safflower and castor,the 33 residue transit peptide is cleaved off to yield a 363 amino acidmature desaturase polypeptide with an apparent molecular weight of 37kDa per subunit by SDS-PAGE. The enzyme occurs as dimers ofapproximately 70 kDa. The enzymatic reaction requires molecular oxygen,NAD(P)H, NAD(P)H ferredoxin oxido-reductase and ferredoxin.

Previous studies have shown that both soluble and membrane-bound Δ⁹desaturases require non-haem iron for catalytic activity. More recently,spectroscopic analysis and amino acid sequence comparisons haveestablished that the Δ⁹ desaturase contains a diiron cluster. This classof diiron proteins is characterized by two occurrences of the sequencemotif E-X-X-H, spaced by approximately 100 amino acids, and includes theR2 subunit of ribonucleotide reductase and a methane monooxygenasehydroxylase component. A greater understanding of the catalyticmechanism of the acyl-ACP desaturase enzymes may enable the exploitationof such enzymes in the manufacture of plant seed oil.

SUMMARY OF THE INVENTION

The subjection invention relates to a method for modifying the chainlength and double bond positional specificities of a soluble plant fattyacid desaturase. More specifically, the method involves modifying aminoacid contact residues in the substrate binding channel of the solublefatty acid desaturase which contact the fatty acid. In preferredembodiments, the soluble plant fatty acid desaturase is an acyl-ACPdesaturase.

Amino acid contact residues which lie within the substrate bindingchannel are identified, for example, by first providing the primaryamino acid sequence of the acyl-ACP desaturase. Many such sequences areknown, and others can be determined through the application of routineexperimentation. Such amino acid sequences are then aligned with theprimary amino acid sequence of the Ricinus communis Δ⁹ desaturase formaximum sequence conservation. A 3-dimensional model for the acyl-ACPdesaturase is then constructed based on the sequence conservation withthe Ricinus communis Δ⁹ desaturase. Amino acid contact residues withinthe substrate binding channel of the modeled structure are thenidentified. A mutant acyl-ACP desaturase having modified chain lengthand double bond positional specificities is then generated by replacingone or more of the amino acid contact residues with another amino acidresidue.

In another aspect, the present invention relates to a mutant acyl-ACPdesaturase which is characterized by the ability to catalyzedesaturation of a first fatty acid and a second fatty acid, the firstand second fatty acids differing in their chain length. This mutant isfurther characterized by the ability to desaturate both the first andsecond fatty acids at rates differing by no more than about 4-fold.

The invention also relates to compositions such as a nucleic acidsequences and expression vectors encoding a mutant acyl-ACP desaturaseof the type described above. Other compositions of the present inventioninclude cells transformed with such expression vectors. In anotheraspect the present invention relates to chimeric acyl-ACP desaturaseshaving modified chain length and double bond positional specificities.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 summarizes the specific activities of wild-type and mutantacyl-ACP desaturases.

FIG. 2 is a linear representation of a chimera of the present inventionand a sequence comparison of amino acids 178-207 of the mature Δ⁶-16:0-(Δ⁶) and Δ⁹ -18:0-(Δ⁹) ACP desaturases from T. alata. Numbers initalics refer to amino acid positions in the mature Δ⁹ -18:0-ACPdesaturase, and non-italicized numbers indicate amino acid positions inthe mature Δ⁶ -16:0-ACP desaturase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the identification of thesubstrate binding groove, and critical contact residues which line thegroove in a Δ⁹ acyl-ACP desaturase. In addition, the invention involvesassaying the effects of logic based site-directed mutations. Prior tothis work, the relative location of the substrate binding channel ofacyl-ACP desaturases, and critical substrate contact residues, had beenunknown.

Acyl-ACP desaturases are highly conserved, with >70% amino acid sequencehomology found between members of different families such as the Δ⁴, Δ⁶and Δ⁹ acyl-ACP desaturases. Each of these desaturases is known tocatalyze the formation of double bonds between carbon atoms of the sameor similar substrate fatty acids. The primary difference between thevarious acyl-ACP desaturase activities is the location of the carbonatoms within the substrate fatty acids that are to be desaturated.

Amino acid sequence conservation is even greater within a particularfamily of acyl-ACP desaturases such as Δ⁹. Based on the presentdisclosure one of skill in the art would predict that contact residueswithin the substrate binding channel of all Δ⁹ -acyl-ACP desaturasemembers are substantially similar, if not identical, to those identifiedin the Δ⁹ -acyl-ACP desaturase described in Example 1 below.

The high degree of amino acid sequence homology within a family ofacyl-ACP desaturases which catalyze the same enzyme reactions, and aminosequence homology between families of acyl-ACP desaturases that catalyzedifferent enzyme reactions suggests that certain portions of the enzymeswill exhibit similar tertiary structures. This is consistent with thefinding for other molecules, such as antibodies, where conservation ofamino acid residue homology is normally greatest within those aminoacids involved in maintaining the functional structure of the moleculeof question.

One such structural region in acyl-ACP desaturases which is conservedbetween the different acyl-ACP desaturases is the substrate bindingchannel described in the Exemplification section which follows. Thesubstrate binding channel described below exhibits an architectureproviding near perfect accommodation for the fatty acid substrate. Ifnot unprecedented, such an exquisite fit is extremely uncommon.

The fact that this substrate binding channel is highly conserved can beconfirmed by aligning for maximum identity (by coventional techniques)the amino acid sequences of members of other acyl-ACP desaturasefamilies with that of the Castor (i.e., Ricinus communis) Δ⁹ acyl-ACPdesaturase described in Example 1 below. The deduced amino acid sequenceof this Castor protein was reported by Shanklin and Somerville (Proc.Natl. Acad. Sci. USA 88: 2510 (1991)). Following this alignment, a3-dimensional model can be generated which will reveal thecharacteristic substrate binding channel. Among the acyl-ACP desaturasesequences from various plant sources determined to date, the followingare available through GenBank (accession codes shown in squarebrackets): [BRSACP] B. rapa; [CAHSACPD] C. tinctorius; [SMMSCPD]Simmondsia chinensis; [SOACCPDS] S. oleracea; [SSMSACPD] sesame plantsource; [TAU07597] Thunbergia alata (clone pTAD2 Δ9); [TAU07605]Thunbergia alata (clone pTAD3 Δ⁹); [ATSTACPDS] A. thaliana; [BNAACPDES]B. napus; [BNSACPD] B. napus; [GHSACPDES] G. hinsutum; [LUSACPDE] L.usitatissimum; [RCSTEA] R. communis; [SOYSACPD] Glycine max; [SSMSACPDA]sesame plant; [SSMSACPDB] sesame plant; [TAU07552] Thunbergia alata(clone pTAD1 Δ⁹).

By studying the results of the molecular modeling for any of theacyl-ACP desaturases, as was done below in connection with the Castor Δ⁹acyl-ACP desaturase, amino acid residues within the substrate bindingchannel which are oriented such that they will be in very closeproximity to the fatty acid substrate can be identified. Such residuesare referred to as "contact residues". As revealed through thedescription of the experimental work below, the modification of acontact residue (and in some cases, other residues as exemplified by thechimeras of Examples 2 and 3) can alter the chain-length and double bondpositional specificities of an acyl-ACP desaturase.

For example, as shown in Examples 2 and 3 below, a chimera was producedwherein amino acids 172-202 of Δ⁶ -16:0-ACP desaturase were replaced byamino acids 178-207 of Δ⁹ -18:0-ACP desaturase. This led to theintroduction of 9 novel amino acids into the substrate binding channelof the Δ⁶ -16:0-ACP desaturase that differed from the amino acids at thecorresponding positions in the wild-type Δ⁶ -16:0-ACP desaturase. Thechimera was not only able to desaturate the 16:0 fatty acid the wildtype functioned best with, but was also able to desaturate an 18:0 atboth the Δ⁶ and Δ⁹ positions at equivalent levels.

The fact that the amino acid contact residues in the substrate bindingchannel of an acyl-ACP desaturase play such a critical role indetermining chain length and double bond positional specificity offersan opportunity for the rational design of mutant acyl-ACP desaturaseswhich have unique and useful properties.

Such novel mutant molecules can be designed, for example, by firstidentifying contact residues within the substrate binding channel (asdescribed above through alignment with the Castor Δ⁹ amino acid sequencefollowed by 3-dimensional modelling). Specific point mutations can thenbe introduced into the acyl-ACP desaturase molecule of interest. This ismost conveniently done at the genetic level using common techniques suchas site-directed mutagenesis.

A variety of site-directed mutagenic techniques can be applied tointroduce a specific amino acid codon change (i.e., substitution) withinsuch DNAs. Care must be exercised in selecting a residue to besubstituted for an existing contact residue in the substrate bindingchannel of a wild-type acyl-ACP desaturase. It is generally important ininitial studies, for example, to select residues for substitution whichdo not differ radically with respect to side chain size or charge. Forexample, if a glycine contact residue (characterized by its compactaliphatic side chain) is identified within the substrate bindingchannel, the substitution of an amino acid residue such as arginine(characterized by the presence of a bulky, basic side chain) could serveto block entry of the fatty acid substrate into the substrate bindingchannel through stearic hindrance. In general, initial amino acidsubstitutions for contact residues should be made using amino acidshaving similar charge characteristics Zenith relatively smalldifferences in terms of side chain bulk. This having been said, it iscertainly possible that the substitution of an amino acid havingradically different properties from a wild-type contact residue mayyield a particularly useful mutant acyl-ACP desaturase. Such a moleculewould be encompassed by the present invention. The brief discussion ofsubstitution strategy given above is intended only to serves as a guideto the incremental modification of an acyl-ACP desaturase.

Thus, it is the knowledge of the identity of the contact residues withinan acyl-ACP desaturase that allow one skilled in the art to makemodifications to the enzyme that can alter the chain-length and doublebond positional specificities of the enzyme without inhibiting itsability to carry out enzyme catalysis. This knowledge, in turn, isdependent upon the ability of one of skill in the art to identify thesubstrate binding channel, and generate a 3-dimensional model.

As already discussed, the nucleotide sequences of many acyl-ACPdesaturase have been reported. Furthermore, given their high degree ofconservation, routine nucleic acid hybridization experiments carried outusing DNA isolated from a plant of interest, would be likely to yieldDNA encoding additional acyl-ACP desaturases.

Further, as indicated above, one of skill in the art would predict thatwithin the Δ⁹ acyl-ACP desaturase family, amino acid contact residueswithin the substrate binding groove would be substantially similar, ifnot identical. The amino acid contact residues identified by the X-raycrystallographic work described in Example 1 are residues M114, L115,T117, L118, P179, T181, G188 and F189. That modification of theseresidues in a Δ⁹ acyl-ACP desaturase does, in fact, alter thechain-length and double bond positional specificies of the enzyme wasconfirmed in the experiments described in Example 2. More specificallypreliminary experimental work has revealed that a single amino acidsubstitution at postion 118 (Leu to Phe) in Castor Δ⁹ acyl-ACPdesaturase results in an approximately 10-fold increase in its activitywith 16:0-ACP. Thus, one amino acid substitution at a contact residueposition can generate an acyl-ACP desaturase with novel and usefulproperties.

Prior to this invention, the only source of variant acyl-ACP desaturaseswas plant tissue which synthesizes unusual isomers of monounsaturatedfatty acids. For example, the Δ⁴ -16:0-ACP desaturase was derived fromseed endosperm of coriander, a tissue that produces large amounts ofpetroselinic acid (18:1Δ⁶), an unusual monounsaturated fatty acid. Thepresent invention enables the design and production of new types ofacyl-ACP desaturases without the need for isolating cDNAs for theseenzymes from plant sources. In addition, the present invention enablesthe design of acyl-ACP desaturases that can catalyze the synthesis ofeconomically valuable monounsaturated fatty acids that are not found innature.

In a more specific example, this invention offers an alternative meansof petroselinic acid production in plants. This fatty acid has a numberof potential industrial and nutritional uses. The only known pathway ofpetroselinic acid formation in plants involves the Δ⁴ desaturation of16:0-ACP followed by elongation of the resulting 16:1Δ⁴ -ACP to form18:1Δ⁶ (or petroselinoyl)-ACP. This pathway requires, among otherthings, a novel acyl-ACP desaturase and a specific acyl-ACP elongationsystem. Among the mutant desaturases described below are enzymes thatcan catalyze the Δ⁶ desaturation of 18:0-ACP to form petrosinoyl-ACP.Such enzymes are useful for the production of petroselinic acid intransgenic crop plants without the need to transfer additional genes forthe 16:1Δ⁴ -ACP elongation pathway. This pathway is a current limitationin efforts to produce petroselinic acid in transgenic crop plantsthrough the introduction of the gene for Δ⁴ -16:0-ACP desaturase.

Thus, mutants generated by altering the identity of one or more contactresidues in the substrate binding channel can be used to generateacyl-ACP desaturases having unique functional characteristics. Suchenzymes can be used, for example, to generate vegetable oils rich inmonounsaturated fatty acids. Such vegetable oils are important in humannutrition and can be used as renewable sources of industrial chemicals.In addition, the ability to manipulate chain length preferences anddouble bond positions of these molecules offers a way to manipulatephysical properties and commecial uses of conventional plant oils. Inaddition, the development of transgenic crops capable of producingunusual types of monounsaturated fatty acids can be exploited based onthe present disclosure.

Mutants disclosed in the Example 2 below exhibit certain uniqueproperties. For example, wild-type acyl-ACP desaturases tend to exhibitvery strong preferences for a particular chain length fatty acid andbond position. However, in the experiments described below, amino acidsubstitutions for contact residues within the substrate binding channelhave been shown to modify this preference. For example, chimeric mutantsare described which exhibit the ability to catalyze desaturation ofsubstrates of different lengths (e.g., 16:0 and 18:0) at rates differingby no more than about 4-fold.

Nucleic acid sequences encoding these mutant acyl-ACP desaturases can beused to express the mutant enzyme using recombinant DNA techniques. Forexample, when cloned in an appropriate expression vector, the mutantacyl-ACP desaturase can be expressed in a variety of cell typesincluding, for example, prokaryotic and eukaryotic cells.

Prokaryotic expression vectors are useful, for example, for thepreparation of large quantities of the protein encoded by the DNAsequence of interest. Following purification by conventional methods,this protein can be used to desaturate a fatty acid. In addition, forsome applications a crude lysate of such a prokaryotic cell culture maybe useful.

Eukaryotic expression vectors are useful when the addition ofcarbohydrate side chains (i.e., glycosylation) to the protein isimportant. The carbohydrate side chains affect the activity of a proteinin several ways. For example, it is known that certain proteins areinactive in their non-glycosylated state. In addition, the ability of anon-glycosylated protein to form a complex with other proteins (e.g.,antibodies or regulatory molecules) can be adversely affected in theabsence of glycosylation. Following purification by conventionalmethods, an acyl-ACP desaturase mutant expressed in a eukaryotic system(e.g., the baculovirus expression system) can be used to modify thechain-length and double bond position of a fatty acid. This protein canalso be used as part of a crude lysate in many circumstances.

The mutant acyl-ACP desaturases can also be cloned into a plantexpression vector. These vectors allow the production of a desiredprotein product, for example the mutant acyl-ACP desaturase, within themilieu of the plant cell within which the substrate fatty acid reside.By producing the enzyme in situ, modification of the product can occurprior to harvest, allowing rapid purification of the desired fatty acidwith the appropriate double bond position, and without the need ofcostly manufacturing steps. In some instances, more than one mutantacyl-ACP desaturase may be desired in a particular transgenic plant toproduce fatty acids with double bonds at multiple positions. Plants arealso easy to cultivate, and grow in large quantity. This protein canalso be used as part of a crude lysate in many circumstances.

EXEMPLIFICATION Example 1 Results and Discussion

Electron density map and quality of the model

The three-dimensional structure of recombinant homodimeric Δ⁹stearoyl-acyl carrier protein desaturase, the archetype of the solubleplant fatty acid desaturases that convert saturated to unsaturated fattyacids, has been determined by protein crystallographic methods to aresolution of 2.4 Å. The six-fold averaged electron density for the mainchain and side chains for most of the polypeptide chain is well defined.Exceptions are the first 18 residues at the N-terminus, which are notdefined in electron density and might be flexible in the crystallattice. Residues 336-347 located in a loop region, are very poorlydefined in the electron density maps and it is also in this part of theprotein structure where the largest deviations from thenoncrystallographic symmetry are found. The overall residue by residuereal space correlation (Br subunit and the six-fold averaged 2Fo-Fcelectron density map is 0.76). Criteria such as crystallographicR-factor (R=22.0%, R_(free) =28.5% with noncrystallographic symmetryrestraints), good stereochemistry of the model (bond length rms of 0.008Å), Ramachandran plot (only one outlier from the allowed regions persubunit, except glycine residues) and the observed hydrogen bondingpattern all indicate that the chain tracing for fatty acid desaturase iscorrect. There is very clear density for the peptide oxygen of Lys 262,the residue with a disallowed main chain conformation. The high averageβ-factor suggest that the molecule is flexible. The most ordered partsof the molecule are areas involved in dimer and hexamer interactionswhereas surface loops often have very high β-factors.

The major binding sites for the Au(CN)₂ --ions in the derivatizeddesaturase crystals are found close to the side chains of K56 and C61 atthe surface of the molecule. One of the minor sites is internal, betweenthe side chains of H203 and C222, and the second minor site is in thearea of where the N-terminus of the chain probably is situated.

The overall shape of the Δ⁹ desaturase subunit is a compact cylinder ofdimensions 35×35×50 with an accessible surface area of 16773. Besides aβ-hairpin-loop at the very C-terminus of the chain, the subunit ismainly composed of helical secondary structures folded into one largedomain. Nine of the total eleven α-helices form an antiparallel helicalbundle.

The N-terminal part of the chain is disordered, no electron density isobserved for the first 18 residues. The next 15 residues lack secondarystructure and form an extended chain packing along the helix bundle withfew specific interactions to stabilize its structure. The first helix,α1, composed of 23 residues, starts and ends in 3₁₀ -conformation and isvery bent so that its first half forms a cap at one end of the bundleand its second part is the first helix of the bundle. The chaincontinues in the same direction forming hydrogen-bonded turns and a 3₁₀-helix. The cap at the other end of the bundle is formed by helices α2and αΔ⁹ and the C-terminal-hairpin. Four of these helices, α3, α4, α6and α7, which are very long, 28, 29, 30 and 31 residues respectively,contribute ligands to the diiron center.

Although α3 has a break in the helical structure in the middle atresidue 107-108, α3 and α4 are symmetrical to α6 and α7 and can besuperimposed with an r.m.s deviation of 1.39 Å for 44 residues. Such asuperposition also aligns the iron atoms to within 1.0 Å. Thecorresponding sequence alignments show that there is little sequenceconservation besides the residues involved in binding the iron cluster.This superposition also orients the cap part of α1 onto α2. Theconnections between these helices also approximately superimposealthough there is no detailed structural similarity. Between α3b and α4there is a protruding loop structure stabilized by severalhydrogen-bonded turns. Helices α5, α8, α10 and α11, which is verycurved, complete the bundle.

There are a large number of salt bridges, 25, excluding thoseinteracting with the iron ions, within the subunit. This corresponds to0.069 ion pairs per residue, higher than the average number of ion pairsper residue, 0.04, derived from a survey of 38 high resolution proteinstructures. Seven of the salt bridges in Δ⁹ desaturase are involved ininter-helix interactions within the bundle, securing the correct mutualpacking and in some instances correct orientation of the iron-ligands.Eight pairs make intra-helix contacts and three of the remaining areinvolved in anchoring the turns between bundle-helices. Three pairs areinvolved in contacts to the 3₁₀ -helix and the loop after α8. A peculiarfeature of the Δ⁹ desaturase subunit is a rather flat surface formed byhelices α1, α6, α7, α10 and α11. This surface is not involved insubunit-subunit contacts in the dimer but is accessible from thesolution.

The Dimer

The subunit-subunit interface in the dimer buries a surface area of 5826Å² per dimer, 17.4% of the dimer area. These two fold interactionsinclude extensive contacts between helices in the bundles; from α3b tothe same helix in the second subunit, between α4 and α5, and over α2 andα4 to the corresponding helices in the other subunit. There are alsomany contacts between the protruding loop between α3b and α4 and theN-terminal, α3b, and α5. In addition, residues in the connection betweenα1 and α2 make contacts to α4 and α5 in the second subunit. There arethree charged interactions in the dimer contact area, two of theseinvolve residues from α5. The diiron centers are separated by more than23 Å in the dimer and have no direct contacts to each other.

Noncrystallographic Symmetry and Crystal Packing

The crystal asymmetric unit contains three Δ⁹ desaturase dimers. Inthese dimers the subunits are related by two-fold noncrystallographicaxes which for one of the dimers is parallel to one of thecrystallographic two fold-axes. At right angles to this, parallel to a,there is a three-fold noncrystallographic screw-axis relating the threedimers. The translation is one third of the length of a, i.e. it is alocal 31 axis. The contacts between the dimers are not extensive, of thesame order as other crystal contacts and the influence of crystalcontacts on the structure seems to be minor as judged from the smalldeviations in non-crystallographic symmetry observed. The largestdeviations are obtained for residues 336-347 where R336, E347 and/orK346 make crystal contacts, including salt bridges, in some of thesubunits. The electrondensity in this area is weak as mentioned above.Another area with deviations from the noncrystallographic symmetryincludes residues 19 to 50 which are wrapped around the subunit and alsoare making different loose crystal contacts in the subunits. The packingof subunits corresponding to one asymmetric unit, viewed along thethree-fold and one of the two-fold axes.

The Diiron Center

Previous studies have shown that Δ⁹ desaturase contains four iron atomsper dimer and optical and Mossbauer spectroscopy indicated that theseiron ions comprise a diiron-oxo-clusters. Diiron-oxo-clusters have nowbeen identified in a wide variety of proteins that perform bothcatalytic and non-catalytic functions. They contain two iron atomsconnected by either an oxo- or hydroxo-bridging ligand and have beenclassified based on differing primary sequence motifs providing thecluster ligands, and upon structural differences elucidated by X-raycrystallography. Four classes have been described, one containinghaemerythrin and myohemerythrin, a second containing the R2 subunit ofribonucleotide reductase, bacterial hydrocarbon hydroxylases, and the Δ⁹desaturase, a third containing rubrerythrin, and a fourth containingFe(III)- Zn(II) purple acid phosphatase (Strmammalian Fe(II)--Fe(II)acid phosphatases). In addition to these soluble proteins, there is adistinct class of functionally related integral membrane proteinsincluding fatty acid desaturases and hydrocarbon hydroxylases whichcontain oxygen-activated non-heme ironcenters, which have yet to bestructurally characterized.

The crystal structure of Δ⁹ desaturase reveals that the enzyme belongsto class II diiron proteins and that it contains a metal cluster. Thedistance between the iron ions is 4.2 Å and the coordination geometry ofthe iron ions is a distorted octahedron where one of ligand positions isunoccupied. The structure of the cluster is highly symmetric. E143 fromα4 and E229 from α7 both act as bridging ligands. E105 from α3a is abidentoate ligand to one iron ion and correspondingly, E196 from α6 is abidentate ligand to the second iron ion. Each iron ion is also ligatedby a nitrogen atom, Nδ1 in H146 from α4 and H232 from α7 respectively.The orientation of the iron ligands is in some cases maintained by sidechain hydrogen bonds; E105 interacts with H203, E143 with atom Nε1 inW139, Nε2 in H146 with the side chain of D228 which in turn interactswith the sidechains of R145 and W62, Nε2 in H232 with the side chain ofE143 which in turn interacts with the side chain of R231. Further awayfrom one of the iron ions. is found atom Nε1 in W139 which might beconsidered to be a second shell ligand. In the vicinity of the ironcluster, there is electron density corresponding to a solvent molecule.Its distances to the iron ions are 3.2 and 3.4 Å, respectively and it istherefore not part of the first coordination shell of the metal center.

Form of the Desaturase in the Crystal Structure

The presence of a μ-oxo bridged diiron cluster in the diferric state ofΔ⁹ desaturase has been unambiguously demonstrated using Mossbauer andresonance Raman spectroscopy. It is therefore surprising, that a μ-oxobridge was not observed in the electron density map of Δ⁹ desaturasebecause the enzyme used for the experiments was in the oxidized stateand noreducing agents were added to the mother liquor. In addition, thedistance between the iron ions (4.2 Å) is longer than expected for adiiron cluster with an intact μ-oxo bridge. In the oxidized form ofribonucleotide reductase with the μ-oxo bridge present, the iron-irondistance is 3.3 Å. The geometry observed in Δ⁹ desaturase is strikinglysimilar to that seen in the reduced form of ribonucleotide reductase,where, upon chemical reduction of R2, the distance between the iron ionsis increased to 3.8 Å, the μ-oxo bridge is lost and the ligandarrangement becomes very symmetric as shown by protein crystallographyand Mossbauer spectroscopy. It can be suggested that exposure of thedesaturase crystals to X-ray radiation results in photochemicalreduction of the metal center which is accompanied by loss of the μ-oxobridge and ligand rearrangement. Thus, the structure of the Δ⁹desaturase presented here most likely represents the reduced form of theenzyme. The crystal structure of the Δ⁹ desaturase reveals a highlysymmetric ligand arrangement of the iron cluster in the diferrous formof the enzyme, in agreement with resonance Raman studies. Deviations inthe symmetric ligand arrangement in the reduced state of the enzyme assuggested from previous temperature dependent Mossbauer data might bedue to variations in bond lengths and bond angles in the two metalsites, too small to be observable in the electron density maps at thecurrent resolution.

Active Site and Interactions with Other Proteins

The structure of Δ⁹ desaturase described here is very likely that of thediferrous form of the enzyme that results from interaction of Δ⁹desaturase and ferredoxin in vivo. From the three-dimensional structure,two possible routes for electrontransfer from the surface to the ironcenter can be postulated. One of these extends along the axis of thehelix bundle and involves the structurally consecutive cluster ofaromatic sidechains of W139, W135, Y236, F189, W132. The Nε1 atom ofW139 is in rather close distance to one of the irons and the Ne1 atom ofW132 points towards the surface of the subunit close to the protrudingloop between α3b and α4. This loop and the cap-part of α1 could thenform a possible interaction surface for the ferredoxin molecule. Anotherpossible route for electron transfer from the surface to the iron centerinvolves residues W62, D228 and H146 analogous to what has beensuggested for R2. This pathway leads to the flat surface formed byhelices α1, α6, α7, α10 and α11.

The solvent molecule bound in the vicinity of the iron center is locatedin a small, hydrophobic pocket and the closest amino acid side chains tothis solvent molecule are T199 and W139. A similar cavity, with a Thrside chain at the equivalent position has been found in MMO and it hasbeen suggested that this cavity could provide a suitable binding sitefor the oxygen molecule. The side chain of T213 in MMO has beenimplicated to be involved in oxygen activation in a similar manner asresidue T252 in cytochrome P450.

Because the iron center is buried in the interior of the Δ⁹ desaturase,a substrate cleft lined with hydrophobic residues connecting the surfaceof the enzyme to the active site was expected to be identified. Indeed,a narrow, very deep channel can be found extending from the surface farinto the protein. The channel passes the diiron center on the same sideas the proposed oxygen binding site. At the bottom of this channel isfound the side chain of L115 and the walls consist of residues W139,T192, Y111, M114, Y191, Q195, P266, T99, and T104. The channel thenpasses the iron cluster and continues towards the surface with residuesY292, M265, F279, and S283 at the narrow entrance of this cleft. Theoverall shape of the substrate channel which is bent at the position ofthe iron cluster facilitates binding of the product, oleoyl-ACP with cisconfiguration at the double bond.

After refinement, strong elongated electron density was found in theaveraged 2Fo-Fc electron density maps in this channel which had not beenassigned to solvent or protein atoms. Based on the shape of this densityand the hydrophobic character of the pocket it can be inferred that thiselectron density may represent the hydrophobic acyl-tail of aβ-octylglucoside molecule. The hydrocarbon tail of the octylglucosidewould fit well in this density but the density corresponding to thesugar moiety is poorly defined. This putative octylglucoside molecule isoriented with its tail deep down in the hydrophobic pocket close to thediiron cluster and the carbohydrate moiety extending towards thesurface. The weak electron density for this part of the molecule mightindicate local disorder resulting from less specific interactions withthe enzyme.

Modeling of a stearic acid in the presumed substrate binding pocketrenders the C9 carbon atom at about 5.5 Å from one of the iron ions.This carbon atom, where the double bond will be formed, is also close tothe small pocket with the bound solvent molecule, in fact the watermolecule is bridging the distance between the C9 carbon of the substrateand the closest iron ion. In the active enzyme, this pocket is likelyoccupied by an oxygen molecule bound to one or both of the iron atoms.During catalysis, a peroxide radical could be generated capable ofabstracting one of the hydrogen atoms at the C9 position of the fattyacid.

Comparison to other Diiron Proteins

A superposition of the structure of Δ⁹ desaturase on thethree-dimensional structures of two other diiron proteins, the R2subunit of ribonucleotide reductase from Escherichia coli and theα-subunit of MMO from Methylococcus capsulate shows that the overallstructures are rather similar, with an r.m.s. fit of 1.90 Å for 144Cα-atoms (Δ⁹ desaturase vs R2) and an r.m.s. fit of 1.98 Å for 117equivalent Cα atoms (Δ⁹ desaturase vs MMO). The folds are very similar,most of the α-helices, α1 to α8 and α10 have their counterpart in R2 andMMO. There are few conserved amino acids besides the iron ligands butthere can be little doubt that these enzymes are evolutionary related.

There are significant differences in the structure of the iron centersin the three proteins. In general, the metal center in Δ⁹ desaturase isconsiderably more symmetrical than in the two other proteins. However,when compared to the structure of the reduced form of R2, thecoordination geometries of the dinuclear iron center in Δ⁹ desaturaseand R2 are more similar. The most significant difference is that in Δ⁹desaturase, the terminal carboxylates E105 and E196, respectively act asbidentate ligands, whereas in R2, the equivalent side chains aremonodentate ligands to the iron ions.

R2 is unique among these enzymes in that it forms a stable radical atposition Y122. The corresponding residue in Δ⁹ desaturase is L150,located in the hydrophobic cluster making packing interactions in thefour-helix bundle binding the iron cluster and no evidence is availablewhich might indicate that this residue is required for catalyticactivity.

There are very few amino acid residues which are conserved in all threeenzymes. Among those are the ligands to the metal ions with theexception of E105 which is replaced by anaspartic acid in R2. The onlyother invariant residues are I225 and D228. Residue I225 is in thevicinity of the diiron cluster (closest distance 4.6 Å) on the oppositeside of the substrate channel. The side chain is packed between H203,H146 and W62 in the three-dimensional structure, and a more detailedexamination of its function has to await the results from site-directedmutagenesis studies. The other invariant protein residue in the threeenzymes, D228, is part of an electron transfer pathway from thedinuclear iron center to the surface of the protein which has beensuggested for R2. In R2, this pathway runs from one of the iron ions viathe side chain of H118, D237 to W48, which is located at the surface ofthe protein. These residues are conserved in Δ⁹ desaturase and a similarpathway for electron transfer can be postulated including thestructurally equivalent residues H146, D228 and W62 as mentioned before.Furthermore, a slightly modified pathway for electron transfer couldalso be suggested for MMO. In this case, the iron ligand (H147) and theaspartic acid residues (D242) are conserved, however the structure atthe surface is different. Nevertheless, an aromatic side chain (Y67) atthe surface is in the vicinity of the side chain of D242.

Most of the other residues conserved between Δ⁹ desaturase and R2 on onehand and Δ⁹ desaturase and MMO on the other hand are located at thesurface of the protein, or involved in packing interactions. Conservedresidues common between R2 and Δ⁹ desaturase in the proximity of thediiron site are residues W135 and W139. While W135 and W139 are strictlyconserved in the desaturases, the corresponding residues W107 and W111in R2 are not strictly conserved. Except for the T4 and E. coli protein,W135 is replaced by a phenylalanine or a tyrosine side chain. Similarly,W139 is replaced by a glutamine residue.

Materials and Methods

Enzyme Purification and Crystallization

Recombinant Δ⁹ desaturase was expressed in E. coli cells and purified asdescribed previously (Fox et al., Biochemistry 33:127766 (1993)).Crystallization of the enzyme was achieved according to (Schneider, etal., J. Mol. Biol. 225:561, (1992)) with slight modifications. Enzymesamples were concentrated to 12-18 mg/ml. A 7.5 ml aliquot of proteinsolution was mixed with the same amount of the reservoir solution,placed on coverslips and allowed to equilibrate over 1 ml of the wellsolution at 20° C. The reservoir solution contained 0.08 M cacodylatebuffer pH 5.4, 200 mM Mg-acetate, 75 mM ammonium sulphate, 2 mM LiCl, 1mM KCl, 0.2% β-octyl glucoside and 12-15% PEG 4000 as precipitant. Thecrystals were orthorhombic, space group P2₁ 2₁ 2₁ with cell dimensionsa=82.2, b=147.8 and c=198.2 Å.

Data Collection and Preparation of Heavy Metal Derivatives

Attempts to prepare heavy metal derivatives by soaking native crystalsof the enzyme with solutions of various heavy metal salts in motherliquor were not very successful. Most soaking experiments resulted incrystal cracking or non-isomorphous crystals and only one useful heavymetal derivative could be prepared by soaking desaturase crystals inmother liquor containing 5 mM KAu(CN)₂ for one week. X-ray data fromnative and derivative crystals were collected on a UCSD multi-wire areadetector system (Hamlin, Methods Enzymol. 114:416, (1985)) at thedepartment of Molecular Biology, Uppsala. Measured frames were processedwith MADNES (Pflugrath, MADNES: Munich area detector NE system, UsersGuide, Cold Spring Harbor Laboratory, N.Y., USA, (1987)). A secondnative data set was collected at beamline X12-C at NSLS, Department ofBiology, Brookhaven National Laboratory. Data frames were collected as10 oscillations using a MAR Research image plate. The data frames wereprocessed with DENZO and scaled with SCALEPACK.

Phase Determination, Model Building and Crystallographic Refinement

Most crystallographic calculations were done using the CCP4 programsuite (Collaborative Computational Project, Number 4, Acta Crystallogr.D50:760, (1994)). The initial crystallographic analysis was carried outwith the data sets collected on the multi-wire detector to 3.1 Åresolution. The difference Patterson map for the gold derivative wasanalyzed using RSPS (Knight, PhD thesis, Swedish University ofAgricultural Sciences, Uppsala 1989). Two sites, related by a strongcross-peak in the difference Patterson map were used for calculation ofdifference Fourier maps and new sites were identified. Finally 6 mainsites and 12 minor sites were found and the heavy metal parameters wererefined using MLPHARE. From results of the rotation functioncalculations and the positions of the metal ions, the direction andposition of the local symmetry operators, relating the six subunits ofΔ⁹ desaturase in the asymmetric unit could be determined. Six-foldnoncrystallographic symmetry averaging using the RAVE program (Jones, inCCP4 Study Weekend 1992: Molecular Replacement (Dodson, E. J., Gover, S.and Wolf, W., eds.) pp. 91-105, Daresbury Laboratory, Daresbury,UK,(1992)) and a spherical envelope, centered at the presumed positionof one Δ⁹ desaturase subunit, was then used to refine the initial SIRphases. From an electron density map at low resolution, based on thesephases, part of the central four-helix bundle, coordinating the diironcenter and the iron atoms could be identified. The coordinates of theiron atoms were refined from the anomalous native data and new phaseswere calculated based on the Au-derivative and the anomalouscontribution from the iron atoms. A new envelope for the subunit wasmade using MAMA (Kleywegt and Jones, Acta Cryst. D50:178 (1994)) byapproximately orienting a subunit of R2 at the correct position for thehelix-bundle.

After noncrystallographic averaging it was possible to build a startingmodel of the desaturase from the electron density map. Cycles of modelbuilding, refinement in XPLOR (Brunger, A., Acta Crystallogr. A45:50,(1989)) (Brunger, A., The X-PLOR manual, Yale University, New Haven,Conn., (1990)), redefinition of the envelope, refinement of the symmetryoperators using IMP (Kleywegt and Jones, Acta Cryst. D50:171, (1994))and averaging were performed until no new electron density appeared inthe averaged maps. At this stage, one more loop which seemed to have adifferent structure in the subunits was built from the 2Fo-Fc-maps.

Crystallographic refinement was carried out with XPLOR, using the Enghand Huber force field (Engh and Huber, Acta Crystallogr. A47:392,(1991)) and noncrystallographic symmetry restraints. Due to the lowresolution (3.1 Å) of the data set, an overall B-value was used. Themodel at this stage had a crystallographic R-factor of 26.7% withsix-fold noncrystallographic symmetry restraints imposed in therefinement. At this stage of the refinement, a new native data set to2.4 Å resolution collected at NSLS became available and refinementcontinued with this data set. The process of refinement was monitored by2.5% of the reflections which were not included in the refinement butwere used to calculate Rfree (Brunger, A., Nature 355:472 (1992)).

Even at the resolution of 2.4 Å the observation to parameter ratio isjust about one and the refinement problem is ill determined. Therefore,during the whole refinement process, noncrystallographic symmetryrestraints were employed in order to avoid over-fitting of thediffraction data. Only those parts of the structure were not restrainedwhich from the averaged electron density maps were judged not to obeythe noncrystallographic symmetry. This includes residues 19-50, 121-122,127-129, 208-212, 241-253, 259-260, 308-319, 336-348 and some sidechains. The electron density for some residues in the region 336-347 isso weak that their positions must be considered arbitrary and theoccupancies for these atoms were therefore set to zero. Overallanisotropic refinement lowered the free R-factor by about 2%. At thisstage, water molecules were added to the model. Individual B-factorswere also refined but restrained by the noncrystallographic symmetry.The final model has a crystallographic R-factor of 22.0% (R free 28.5%).The r.m.s. deviations for the restrained Cα positions (263 atoms) of thesubunit A to the corresponding parts of the other subunits are 0.06 andfor all Cα atoms (345 atoms) 0.26, 0.23, 0.24, 0.32, 0.25, respectively.

The protein model was analyzed using the PEPFLIP and RSFIT options in O(Jones et al., Acta crystalllogr. A47:100, (1991)) and with the programPROCHECK (Laskowski et al., J. Appl. Crystallogr. 26:282, (1993)). Theatomic coordinates will be deposited with the Brookhaven Protein DataBank.

Structural Comparisons

All structural superpositions were performed by least-squares methodsusing O (Jones et al., Acta crystalllogr. A47:100, (1991)) and were donepair wise. Superposition was done by selecting an initial set ofequivalent Cα atoms consisting of four stretches of the polypeptidechain (about 10 residues each) from the four helices containing theligands to the diiron center. This initial alignment was subsequentlymaximized by including all Cα atoms from the atomic models. Residueswere considered structurally equivalent if they were within 3.8 fromeach other and within a consecutive stretch of more than threeequivalent residues.

Example 2 Results and Discussion

The approach of combining amino acid sequence elements from structurallyrelated enzymes with different properties has proven effective incharacterizing the substrate and positional specificities of fatty acidmodifying enzymes such as mammalian lipoxygenases and plant acyl-ACPthioesterases. This approach was used here to identify the residuesresponsible for the differences in properties of a Δ⁹ -18:0-ACPdesaturase and a Δ⁶ -16:0-ACP desaturase encoded by the T. alata cDNAspTAD2 and pTAΔ⁴, respectively. The mature polypetides encoded by thesecDNAs share 65% amino acid sequence identity. Initially two chimericmutants were constructed: (a) a first chimera contained the first 171amino acids of the mature Δ⁶ -16:0-ACP desaturase linked to theremaining 185 amino acids of the Δ⁹ -18:0-ACP desaturase and (b) asecond chimera contained the first 227 amino acids of the mature Δ⁹-18:0-ACP desaturase linked to the remaining 134 amino acids of the Δ⁶-16:0-ACP desaturase. Both enzymes displayed only detectable Δ⁹-18:0-ACP desaturase activity. In addition to catalyzing a similaractivity, these mulants share a 50 residue region of overlap (residues178-227) of the Δ⁹ -18:0-ACP desaturase.

This suggested that determinants of chain-length and double bondpositional specificities are present in this portion of the Δ⁹ -18:0-ACPdesaturase. Thus, a third chimera was constructed in which residues172-221 of the Δ⁶ -16:0-ACP desaturase were replaced with thecorresponding 50 amino acid region from the Δ⁹ -18:0-ACP desaturase. Theresulting enzyme catalyzed the Δ⁶ or Δ⁹ desaturation of both 16:0-ACPand 18:0-ACP. A nearly identical activity was obtained for a fourthchimera, in which a 30 amino acid subset of this domain (residues178-207 of the Δ⁹ -18:0-ACP desaturase) was transposed into the Δ⁶-16:0-ACP desaturase. As shown in FIG. 1, in sharp contrast to theactivity of the wild-type Δ⁶ -16:0-ACP desaturase this enzyme catalyzedΔ⁶ and Δ⁹ desaturation at a ratio of nearly 3:1 and 1:1 with 16:0-ACPand 18:0-ACP, respectively. Moreover, the specific activity with18:0-ACP as a substrate was nearly twice that detected with 16:0-ACP.These results are in sharp contrast to the activity of the wild-type Δ⁶-16:0-ACP desaturase. Though this chimeric enzyme is able to catalyzethe insertion of a double bond at more than one position of 18:0-ACP,while the wild-type Δ⁶ -16:0-ACP desaturase only has detectable Δ⁶desaturase activity with 16:0-ACP. In addition, the wild-type enzyme wasabout 6-fold more active with 16:0-ACP than with 18:0-ACP.

To further characterize the 50 amino acid region of the Δ⁹ -18:0-ACPdesaturase, a smaller portion of this sequence (residues 178-202) wastransposed into the Δ⁶ -16:0-ACP desaturase (a fifth chimera). Unlikethat of the wild-type Δ⁶ -16:0-ACP desaturase, the specific activity ofthe resulting enzyme was nearly equal with 16:0- and 18:0-ACP. Inaddition to a broadened fatty acid chain-length specificity, the mutantdesaturase catalyzed the insertion of a double bond almost exclusivelyat the Δ⁶ position of 16:0- and 18:0-ACP. Furthermore, the specificactivity of this enzyme was more than two-fold greater than that of thewild-type Δ⁶ -16:0-ACP desaturase. This may in part reflect the greaterstability of the mutant enzyme in E. coli (i.e., the mutant desaturasewas expressed to higher levels and displayed greater solubility than thewild-type Δ⁶ -16:0-ACP desaturase).

Region 178-207 of the Δ⁹ -18:0-ACP desaturase contains nine amino acidsthat are different from those found in the analogous portion of the Δ⁶-16:0-ACP desaturase. Through site-directed mutagenesis of the Δ⁶-16:0-ACP desaturase, each of these residues, either individually or incombination, was converted to that present in the Δ⁹ -18:0-ACPdesaturase. An activity qualitatively similar to that of the fourthchimera was obtained by the following mutation of the Δ⁶ -16:0-ACPdesaturase: A181T/A188G/Y189F/S205N/L206T/G207A. (Note: Amino acidnumbering is given with respect to the Δ⁹ -18:0-ACP desaturase.) Inaddition, the fifth chimera phenotype (i.e., broadened chain-lengthspecificity) was achieved qualitatively by the mutation A188G/Y189F ofthe Δ⁶ -16:0-ACP desaturase. Mutant desaturases with unexpectedactivities were also obtained in these experiments. For example, themutation A181T/A200F of the Δ⁶ -16:0-ACP desaturase gave rise to anenzyme that catalyzed primarily the Δ⁹ desaturation of 18:0-ACP, butfunctioned as a Δ⁶ desaturase with 16:0-ACP. The specific activity ofthis enzyme with 18:0-ACP, however, was about 3-fold less than thatdetected with 16:0-ACP. Furthermore, the mutationA181T/A200F/S205N/L206T/G207A of the Δ⁶ -16:0-ACP desaturase yielded anenzyme that possessed only detectable Δ⁹ desaturase activity with18:0-ACP and was nearly four-fold more active with this substrate thanwith 16:0-ACP. Like mutant A181T/A200F, this enzyme retained Δ⁶desaturase activity with 16:0-ACP.

Changes in the substrate binding properties of these enzymes can bediscounted as an underlying cause of the observed effects because theirvalues are not significantly different from those of the wild typeenzyme. The Km values for the wild-type Δ⁶ -16:0-ACP desaturase, thefifth chimera, and mutant 188G/Y189F were estimated to be in the rangeof 0.2 to 0.6 μM for both 16:0- and 18:0-ACP.

As described in Example 1, the crystal structure of castor Δ⁹ -18:0-ACPdesaturase was determined, making it possible to interpret the resultson chimeras and mutants in light of the arrangement of the active site.The subunit structure contains a very deep and narrow channel whichappears to correspond to the binding site for the stearic acid part ofthe substrate. The form of the channel imposes a bent conformation ofthe aliphatic chain at the point where the double bond is introduced,between carbon 9 and 10, corresponding to the cis configuration of theoleic acid product, positioning the potential double bond rather closeto the catalytic iron center in the subunit. This substrate bindingchannel thus sets severe restrictions on the length of the aliphaticchain beyond the introduced double bond which can in part explain thedifferences in specificity for the enzymes in this family. As can beseen, variants of the enzyme which accept substrates with fewer carbonatoms beyond the double bond, have their binding clefts closed bysubstitutions of amino acid with bulkier side chains. The amino acidsinvolved in determining the specificity in this part of the binding siteare 114-115, 117-118, 179, 181 and 188-189.

In the absence of a structural model for the enzyme-substrate-ACPcomplex, the determinants of chain length specificities on the otherside of the double bond, towards the acyl carrier protein are not asstraightforward to deduce. Assuming that ACP binds in the same way inthe different enzymes of this kind, differences in the amino acid sidechains in the upper part of the substrate channel and at the entrance atthe surface of the subunit would allow the enzymes to accommodatedifferent lengths of the alkyl chain between the double bond and thephosphopantheine prosthetic group of ACP. However, the amino acidslining the upper part of the binding site, from the double bond to thesurface of the protein are conserved in the available enzyme sequencesand determinats for specificity are most likely to be found at theentrance of the substrate channel and at the enzyme surface whichinteracts with acyl-ACP. Here the binding pocket widens and it has notbeen possible to model the phosphopantheine part the stearoyl-ACP.Residues 280, 283, 286 and 294 in this area are not conserved betweenthe different enzymes and might be involved in determining the substratespecificity.

From the structure of the binding site in this area it is possible torationalize some of the results on chimeras and mutants. All thechimeras and mutants involve the determinant 179-189 (actually residues179, 181, 188-189) and it is thus not surprising to find effects onspecificity. Both the first and second chimeras have very littleresidual activity, probably due to some steric clashes upon theirformation. The first chimera has this determinant of Δ⁹ -18:0 ACPdesaturase in the deep pocket and also the surface determinant specificfor of Δ⁹ -18:0 ACP desaturase, only one determinant, residues 114-115and 117-118 specific for of Δ⁶ -16:0 ACP desaturase and thus the littleremaining activity of this chimera is that of a Δ⁹ -18:0 ACP desaturase.The second chimera has the whole determinant of Δ⁹ -18:0 ACP desaturasein the area of the buried pocket and the known determinant of Δ⁶ -16:0ACP desaturase at the surface end; this chimera also has Δ⁹ -18:0 ACPactivity. The third and forth chimeras have retained their activity, oneof the determinants in the deep pocket is that for a Δ⁹ -18:0 ACP,residue A181 is substituted for the larger threonine sidechain but atthe same time A188 is substituted for glycine and Y189 forphenylalanine, actually making more space available in the deep cavityand thus allowing even for Δ⁶ -18:0 ACP activity. The fifth chimeradiffers from the fourth chimera only in that it has retained the Δ⁶-16:0 ACP desaturase sequence for residues 203-207. These residues areat the upper part of the substrate channel but do not make directcontact to the substrates and it is difficult to understand the effecton the substrate specificity. These residues are fairly conservedbetween the known desaturases in this family, only Δ⁶ -16:0 ACPdesaturase has a different sequence for residue 205 to 207, and thisregion probably does not constitute part of the natural determinant forsubstrate specificity. In the case of mutant A181T/A200F the decrease inthe Δ⁶ -16:0 ACP activity compared to the wild type enzyme is consistentwith the structural changes in the substrate channel due to a decreasein size of this cavity by changing A181 to threonine. The effect ofA200F is not possible to rationalize, this residue is on the surface ofthe subunit pointing away from the substrate-channel. In all sequenceddesaturases in this family except Δ⁶ -16:0 ACP this residue is aphenylalanine. From the foregoing discussion it is clear that theactivity of A181T/A200F/S205N/L206T/G207A is impossible to explain instructural terms, we can not rationalize the effects of changes atresidues 200 and 205-207.

Thus, it has been shown that regio- and chain length specificities offatty acid desaturase can be changed by specific amino acidreplacements. The determinants for chain length specificity partly maponto the region of the three-dimensional structure which define shapeand size of the substrate binding channel. However, some of theseresidues lie outside the substrate binding channel and changes in suchresidues may result in new and useful activities. With the availabilityof the three-dimensional structure of fatty acid ACP desaturase, thesuccessful attempts to change the substrate specificities presented herecan now be extended to rationally designed variants of the enzymepossessing different chain length- as well as regio-specificities.However, this will be successful only if we, from the crystal structureof a substrate complex and the availability of multiple amino acidsequences of enzymes in this family, can resolve what are thedeterminants for specificity at the entrance of the substrate channel.

Materials and Methods

Fatty acid names are abbreviated in the format x:ydz where x is thechain-length or numbers of carbon atoms in the fatty acid, y is thenumber of double bonds, and z is the position of the double bond in thefatty acid relative to the carboxyl end of the molecule (e.g., oleicacid or 18:1Δ⁹ is an 18 carbon fatty acid with one double bond, which ispositioned at the ninth carbon atom relative to the carboxyl end of themolecule).

Preparation of Chimeric Mutants

Chimeric mutants were prepared by linking portions of the coding sequnceof the mature T. alata Δ⁶ -16:0- and Δ⁹ -18:0-ACP desaturases via nativerestriction enzyme sites or restriction enzyme sites generated by PCR.Site-specific mutations in the coding sequence of amino acids 178-202 ofthe Δ⁹ -18:0-ACP desaturase (equivalent to residues 172-196 of the Δ⁶-16:0-ACP desaturase) were introduced by extension and amplification ofoverlapping oligonucleotide primers using PCR. with Pfu polymerase(Stratagene). Mutations A181T/A188G/Y189F were made with the followingoligonucleotides: 5'ATGGATCCTGGCACGGATAACAACCCGTAC3' ---SEQ ID NO: 1---(Primer 1A); 5'ACGAGGTGTAGATAAATCCGAGGTACGGGTTGTTATCCG3' ---SEQ ID NO:2--- (Primer 2A); 5'TATCTACACCT-CGTATCAGGAGAGGGCGACA3' ---SEQ ID NO:3--- (Primer 3A); 5'TTGAATTCCATGGGAAATCGCTGTCGCCCTCTCCTG3' ---SEQ ID NO:4--- (Primer 4A). Mutations A188G/Y189F were introduced using thefollowing oligonucleotides: 5'ATGGATCCTGGCGCGGATAACAACCCGTAC3' ---SEQ IDNO: 5--- (Primer 1B); Primer 2A; Primer 3A; Primer 4A. MutationsA181T/A200F were generated with the following: Primer 1A;5'ACGAGGTGTAGATATATGCGAGGTACGG-GTTGTTATCCG3' ---SEQ ID NO: 6--- (Primer2B); Primer 3A; 5'TTGAATTCCATGGGAAATGAATGTCGCCCTCTCCTG3' ---SEQ ID NO:7--- (Primer 4B). PCR reactions were conducted without added templateusing 12.5 pmoles of Primers 1A or B and 4A or B and 6.25 pmoles ofPrimers 2A or B and 3A. For the first 10 PCR cycles, an annealingtemperature of 37° C. and an extension temperature of 72° C. were used.This was followed by an additional 20 cycles with the annealingtemperature increased to 55° C. Products from PCR reactions weredigested with BamHI and EcoRI and inserted into the corresponding sitesof pBluescript II KS(-) (Stratagene) from which the nucleotide sequencewas determined using a Sequenase 2.0 kit (Amersham). This plasmid wasthen digested with BamHI and EcoRI and the recovered insert was ligatedto the coding sequence of amino acids 1-171 of the mature Δ⁶ -16:0-ACPdesaturase in the expression vector pET3a (Novagen). The resultingconstruct (now containing the coding sequnce of amino acids 1-196 ofmutant or wild-type Δ⁶ -16:0-ACP desaturase) was restricted with NcoIand EcoRI and ligated to an NcoI/EcoRI fragment containing the codingsequence of the remaining amino acids (residues 197-355) of the Δ⁶-16:0-ACP desaturase and a portion of the pET3d plasmid (nucleotides).The mutation S205N/L206T/G207A was generated by PCR amplification of thecoding sequence of amino acids 197-355 of the Δ⁶ -16:0-ACP desaturaseusing as template the original cDNA for this enzyme in pBluescriptSK(-). The 5' oligonucleotide(5'TTTCCATGGGAACACGGCTCGGCTAGCGAGGCAGAAGG3' ---SEQ ID NO: 8---),contained the appropriate mutant codons, and the T7 primer was used asthe 3' oligonucleotide for PCR reactions. The amplification product wasdigested with NcoI and BclI and inserted into the NcoI/BamHI site of pET3d. A NcoI/EcoRI fragment from this construct was then ligated to thecoding sequence of amino acids 1-196 of the appropriate mutant Δ⁶-16:0-ACP desaturase (e.g. A181T/A200F) to generate a full-length codingsequence. Products of PCR reactions were sequenced to confirm thepresence of desired mutations.

Production of Acyl-ACP Desaturases

Wild-type and mutant acyl-ACP desaturases were obtained by expression ofthe coding sequences in E. coli BL21 (DE3) behind the T7 RNA polymerasepromoter using the vectors pET3a or pET3d. Recombinant enzymes whoseactivities are described in FIG. 1 were purified from 6 to 9 literbacterial cultures induced at 20 to 25° C. Protein purification wasperformed using DEAE-anionic exchange chromatography followed by 20HS(Perseptive Biosystems) cationic exchange chromatography using a BiocadSprint HPLC (Perseptive Biosystems). Mutant desaturases were obtained atr 90% purity, and the wild-type Δ⁶ -16:0-ACP desaturase was recovered atapproximately 80% purity. Following purification, enzymes were exchangedinto a buffer consisting of 40 mM Tris-HCl (pH 7.5), 40 mM NaCl, and 10%glycerol and stored in aliquots at -75° C. after flash-freezing inliquid nitrogen.

Assay and Analysis of Acyl-ACP Desaturases

Acyl-ACP desaturation assays and analysis of reaction products wereconducted as previously described (Cahoon, E. B., et al. . Proc. Nat.Acad. Sci., USA. 89:1184, (1994)) with the following modifications:recombinant Anaebena vegetative ferredoxin (22 fg/assay) and maize rootFNR (0.4 U/assay) were used in place of spinach ferredoxin and FNR andamounts of NADPH and [1-14C}16:0- or 18:0-ACP per assay were increasedto 2.5 mM and 178 pmoles (or 1.2 fM), respectively. ACP used in thesynthesis of substrates was recombinant spinach ACP-I. The specificactivity of [1-14C}16:0 and 18:0 (American Radiolabeled Chemicals) was55 mCi/mmol. Enzyme activity was measured by determining the percentageof monounsaturated product generated in desaturation assays. Thedistribution of radioactivity between products and unreacted substratewas measured from phosphorimages of argentation TLC separations usingImageQuant software and by liquid scintillation counting of TLCscrapings.

Determination of Double Bond Positions

Double bond positions of monounsaturated fatty acid products weredetermined by the mobility of methyl ester derivatives on 15%argentation TLC plates and by GC-MS analysis of dimethyl disulfideadducts of these derivatives. Desaturation assays for GC-MS analyseswere conducted using unlabelled 16:0-, 17:0-, and 18:0-ACP as substratesand purified enzymes. In addition to results presented in the text,about 15% of the desaturation products formed by the reaction of17:0-ACP with the wild-type Δ⁶ -16:0-ACP desaturase was detected as the17:1Δ⁷ isomer. The remainder of the product was 17:1Δ⁶ with traceamounts of 17:1Δ⁹ also detected.

Example 3 Results and Discussion

Mutant Acyl-ACP Desaturases with Altered Activities

The studies described here were initiated prior to the availability ofthree-dimensional data from the crystal structure of the castor Δ⁹-18:0-ACP desaturase (Lindqvist et al., (1996) EMBO J. 15, 4081-4092).In the absence of this information, efforts were made to understand thestructural basis for differences in activities catalyzed by a Δ⁶-16:0-ACP desaturase and a Δ⁹ -18:0-ACP desaturase by generating aseries of chimeric enzymes that combined portions of these twodesaturases (e.g., see FIG. 2). The activities of the resulting enzymeswere then assessed to determine the effect on their substrate andregio-specifities. In this manner, a 30-amino acid domain encompassingresidues 178-207 of the Δ⁹ -18:0-ACP desaturase was identified thatcontained determinants of both chain-length and double bond positionalspecificities. When this domain was introduced in place of the analogousportion of the Δ⁶ -16:0-ACP desaturase (FIG. 2), the resulting enzyme(designated Chimera 1 of Example 3) displayed a mixture of Δ⁶ and Δ⁹desaturase activities with 16:0- and 18:0-ACP (Table 1). In sharpcontrast to the wild type Δ⁶ -16:0-ACP desaturase, Chimera 1 of Example3 catalyzed Δ⁶ and Δ⁹ desaturation with 16:0-ACP at a ratio of 3:1 andwith 18:0-ACP at a ratio of 1:1 (Table 1). In addition, the specificactivity of Chimera 1 of Example 3 with 18:0-ACP was twice that detectedwith 16:0-ACP (Table 1).

It should be noted that the chimeras discussed in Example 3 were alsodescribed in Example 2. The chimera numbers assigned in theExemplification section are example-specific. Thus, for example, chimera1 of Example 3 is referred to as "a fourth chimera" in Example 2.

                                      TABLE 1                                     __________________________________________________________________________    Specific activites of wild type and mutant Δ.sup.6 -16:0-ACP            desaturases with 16:0- or 18:0-ACP*                                                         Specific activity (nmole/min/mg protein)                                                                  Ratio of Total                                    16:0-ACP      18:0-ACP      Specific Activity                   Enzyme*       Δ.sup.6                                                                          Δ.sup.9                                                                      Δ.sup.6                                                                          Δ.sup.9                                                                      16:0-ACP:18:0-ACP                   __________________________________________________________________________    Δ.sup.6 -16:0-ACP Desaturase                                                          100 ± 3                                                                             n.d..sup.†                                                                  11 ± 1                                                                              5.5 ± 0.2                                                                       6:1                                 (wild type)                      (2:1.sup.‡)                       Chimera 1      13      4.0   15      17   1:2                                 (Δ.sup.6 /Δ.sup.9.sub.178-207 /Δ.sup.6)                                        (3:1)         (1:1)                                        Chimera 2     227 ± 25                                                                            ND   253 ± 25                                                                            5.3 ± 0.5                                                                       1:1                                 (Δ.sup.6 /Δ.sup.9.sub.178-202 /Δ.sup.6)                                                      (50:1)                                       A181T/A188G/Y189F/S205N/                                                                    33 ± 4                                                                              12 ± 1                                                                          31 ± 2                                                                              53 ± 5                                                                          1:2                                 L206T/G207A.sup.§                                                                           (3:1)         (1:2)                                        A188G/Y189F   34 ± 3                                                                              ND   34 ± 4                                                                              0.7 ± 0.1                                                                       1:1                                                                  (50:1)                                       A181T/A200F   149      ND   3.1      44   3:1                                                                  (1:15)                                       A181T/A200F/S205N/L206T/                                                                    19 ± 1                                                                              1.4 ± 0.3                                                                       ND       76 ± 5                                                                          1:4                                 G207A              (14:1)                                                     __________________________________________________________________________     Shown are Δ.sup.6 and Δ.sup.9 desaturase activities with each     substrate ± S.D. of three independent measurements.                        *In addition to results presented in Table 1, approximately 15% of the        desaturation products formed by the reaction of 17:0ACP with the wild typ     Δ.sup.616:0- ACP desaturase was detected by GCMS as the 17:1            Δ.sup.7 isomer. The remainder of the product was 17:1 Δ.sup.6     with trace amounts of 17:1 Δ.sup.9 also detected. Small amounts of      Δ.sup.7 isomers were also present in mass spectra of products forme     from 16:0 and 18:0ACP with the wild type Δ.sup.616:0-ACP                #desaturase and several of the mutants. These accounted for <5% of the        total products. No Δ.sup.8 isomers were detected in mass spectra of     desaturation products.                                                        .sup.† n.d. = not detected.                                            .sup.‡ x:y is the ratio of Δ.sup.6 :Δ.sup.9            activity with the given substrate.                                            .sup.§ Amino acid numbering corresponds to the sequence of the matur     Δ.sup.918:0-ACP desaturase.                                        

An additional novel activity was obtained using a smaller portion of the30-amino acid domain described above. When residues 178-202 of the Δ⁹-18:0-ACP desaturase were introduced in place of the correspondingportion of the Δ⁶ -16:0-ACP desaturase, an enzyme that functioned almostexclusively as a Δ⁶ desaturase with 16:0- and 18:0-ACP was obtained(designated Chimera 2 of Example 3; Table 1). The double bond positionalspecificity with 18:0-ACP was distinctly different than that of the wildtype Δ⁶ -16:0-ACP desaturase which displayed mixed Δ⁶ and Δ⁹ desaturaseactivity with this substrate. In addition, Chimera 2 of Example 3,unlike the wild type enzyme, was almost equally active with 16:0- and18:0-ACP (Table 1). Interestingly, the specific activity of Chimera 2 ofExample 3 with 16:0-ACP was two-fold greater than that detected with thewild type Δ⁶ -16:0-ACP desaturase. Also, the specific activity with18:0-ACP was more than 15-fold greater than that displayed by the wildtype enzyme.

Amino acids 178-207 of the Δ⁹ -18:0-ACP desaturase contain nine residuesthat are different from those found in the equivalent portion of the Δ⁶-16:0-ACP desaturase (FIG. 2). Through site-directed mutagenesis of theΔ⁶ -16:0-ACP desaturase, each of these residues, either individually orin combination, was converted to that present in the Δ⁹ -18:0-ACPdesaturase. An activity qualitatively similar to that of Chimera 1 ofExample 3 was obtained by the following mutation of the Δ⁶ -16:0-ACPdesaturase: A181T/A188G/Y189F/S205N/L206T/G207A (Table 1). In addition,the Chimera 2 of Example 3 phenotype (i.e., broadened fatty acidchain-length specificity) was achieved qualitatively by the mutationA188G/Y189F of the Δ⁶ -16:0-ACP desaturase (Table 1).

Mutant desaturases with unexpected activities were also generated inthese experiments. For example, the mutation A181T/A200F of the Δ⁶-16:0-ACP desaturase gave rise to an enzyme that catalyzed primarily theΔ⁹ desaturation of 18:0-ACP, but functioned as a Δ⁶ desaturase with16:0-ACP (Table 1). Furthermore, the mutationA181T/A200F/S205N/L206T/G207A of the Δ⁶ -16:0-ACP desaturase yielded anenzyme that functioned primarily as a Δ⁹ -18:0-ACP desaturase. Thisenzyme displayed only Δ⁹ desaturase activity with 18:0-ACP and wasnearly four-fold more active with this substrate than with 16:0-ACP(Table 1). However, like mutant A181T/A200F, this enzyme retained Δ⁶desaturase activity with 16:0-ACP.

Of note, the enzyme assay used in these studies precluded the collectionof large amounts of kinetic data. However, K_(m) values determined forthe wild type Δ⁶ -16:0-ACP desaturase, Chimera 2 of Example 3, andmutant A188G/Y189F were in the range of 0.2 to 0.6 μM with both 16:0-and 18:0-ACP, suggesting that, at least in the case of these enzymes,changes in the substrate binding properties can be discounted as anunderlying cause of differences in activities.

Interpretation of Mutant Enzymes in Terms of the Crystal Structure ofthe Δ⁹ -18:0-ACP Desaturase

Recently, the crystal structure of castor Δ⁹ -18:0-ACP desaturase wasdetermined (Lindqvist et al., (1996) EMBO J. 15, 4081-4092), making itpossible to interpret a portion of the above results in terms of thearrangement of the active site. The subunit structure of the Δ⁹-18:0-ACP desaturase contains a catalytic di-iron cluster, whichrepresents a fixed point for the introduction of the double bond.Adjacent to the iron atoms is a deep and narrow channel that likelyrepresents the binding pocket for the stearic acid (18:0) portion of thesubstrate. The channel imposes a bent conformation on the fatty acidchain between carbon atoms 9 and 10, the site of desaturation. Thisconformation corresponds to the cis configuration of the oleoyl(18:1Δ⁹)-ACP product and positions the location of double bond insertionclose to the di-iron center. In this model, the architecture of thefatty acid binding channel in relation to the catalytic iron clusteraffects both substrate specificity and the position at which doublebonds are introduced.

With regard to substrate specificity of acyl-ACP desaturases, thegeometry of the lower portion of the binding pocket places severeconstraints on the length of the acyl chain that can be accommodatedbeyond the point where the double is to be introduced. As such, residuesat the bottom of the substrate channel are likely the most critical fordetermining fatty acid chain-length specificity. This explains thefunctional properties of Chimera 2 of Example 3 and mutant A188G/Y189Fwhich display increased Δ⁶ desaturase activity with 18:0-ACP compared tothe wild type Δ⁶ -16:0-ACP desaturase. Based on the model of the activesite, substitution of alanine 188 for a smaller glycine and tyrosine 189for phenylalanine extends the cavity at the bottom of the active siteenough to accommodate the two additional carbon atoms at the methyl endof 18:0-ACP. As a result, Chimera 2 of Example 3 and mutant A188G/Y189Fare able desaturate 18:0-ACP with a rate comparable to that observedwith 16:0-ACP.

More difficult to interpret are alterations in the double bondpositional specificities of mutants described above. This class of fattyacid desaturases inserts a double bonds at a characteristic positionfrom the carboxyl end of acyl chains (Cahoon and Ohlrogge, (1994) PlantPhysiol. 104, 827-844; and Gibson, (1993) Biochim. Biophys. Acta 1169,231-235). Thus, the site of double bond insertion by acyl-ACPdesaturases is likely associated with interactions between the upperpart of the active site and the ACP portion of substrates. Assuming thatACP binds in the same manner in all acyl-ACP desaturases, differences inthe amino acid side chains at the enzyme surface and/or those lining theupper portion of the substrate binding channel would allow the enzyme toaccommodate different lengths of the fatty acid chain between the siteof double bond insertion and the thioester linkage to ACP. However, inthe absence of a crystal structure of the desaturase that includes abound acyl-ACP substrate, it is not possible to interpret differences indouble bond positional specifities of the mutants described above or innaturally occurring enzymes such as the Δ⁴ - and the Δ⁶ -16:0-ACPdesaturases. Of the residues that contribute to altered double bondpositional specificity in the mutants described above, alanine 200 is onthe surface pointing away from the substrate channel and amino acids205-207 are located outside of the active site. It is possible thatthese residues mediate changes in activity via subtle packing effects inthe enzyme rather than through direct interactions with substrates, ashas been reported for mutants of serine proteases with altered function(Hedstrom, (1994) Curr. Opin. Struct. Biol. 4, 608-611; andSchellenberger et al., (1993) Biochemistry 32, 4349-4353).

Modelling of the Active Sites of Variant Acyl-ACP Desaturases

The crystallographic model of the active site of the castor Δ⁹ -18:0-ACPdesaturase also provides useful information regarding the fatty acidchain-length specifities of naturally-occurring variant acyl-ACPdesaturases (e.g., the Δ⁹ -14:0-, Δ⁴ -16:0-, and Δ⁶ -16::0-ACPdesaturases). Variant desaturases that accept substrates with fewercarbon atoms in the distal portion of the active site (i.e., the regionbeyond the site of double insertion) have their binding clefts occludedby substitutions of amino acids with bulkier side chains. The aminoacids involved in determining the specificity in this part of thebinding site are: 114-115, 117-118, 179, 181, and 188-189. For example,the Δ⁹ -18:0-ACP desaturase, which accepts nine carbon atoms of thefatty acid substrate beyond the point of double bond insertion, containsa methionine, proline, and glycine at amino acid positions 114, 179, and188, respectively. In contrast, the Δ⁹ -14:0-ACP desaturase, whichaccepts five carbon atoms of the substrate in the deep portion of theactive site, contains the larger hydrophobic residues leucine,isoleucine, and leucine at positions 114, 179, and 188, respectively.Such amino acid differences likely contribute to a smaller bindingpocket in the Δ⁹ -14:0-ACP desaturase relative to the Δ⁹ -18:0-ACPdesaturase.

Rational Design of Acyl-ACP Desaturase Activities

Residues described above that line the lower portion of the active siterepresent potential targets for the rational design of acyl-ACPdesaturase activities with respect to fatty acid chain-lengthspecificity. As a test of this, leucine 118 and proline 179 of themature castor Δ⁹ -18:0-ACP desaturase were replaced with the bulkierresidues phenylalanine and isoleucine, respectively. Consistent with theproposed model of the active site, these substitutions yielded an enzymethat was functionally converted from a Δ⁹ -18:0-ACP desaturase to a Δ⁹-16:0-ACP desaturase. The altered activity resulted both from areduction in the specific activity with 18:0-ACP and an increase in thespecific activity with 16:0-ACP. Overall, the specific activity of theL118F/P179I mutant with 16:0-ACP was more than 15-fold greater than thatdisplayed by the wild type Δ⁹ -18:0-ACP desaturase with this substrate.GC-MS analyses of desaturation products indicated that the Δ⁹ positionwas the exclusive site of double bond insertion when the mutant waspresented with 16:0-ACP. However, when 18:0-ACP was provided as asubstrate, not only was 18:1Δ⁹ formed, but approximately 5% of thedesaturation products were detected as the Δ¹⁰ isomer. This isconsistent with a reduced ability of the binding pocket of theL118F/P179I mutant to accommodate the longer acyl chain of the 18:0-ACPsubstrate.

Overall, these results demonstrate the ability to modify the substrateand double bond positional specificities of acyl-ACP desaturases usingamino acid sequence alignments either alone or in conjunction withthree-dimensional structural data from the castor Δ⁹ -18:0-ACPdesaturase. These two approaches provided unique and complementaryinformation with regard to understanding the molecular mechanisms of thesubstrate and regio-specificities displayed by acyl-ACP desaturases. Inaddition, several of the mutants described here have possiblebiotechnological applications. For example, Chimera 2 of Example 3 orthe Δ⁶ -16:0-ACP desaturase mutant A188G/Y189F provides a means ofproducing petroselinic acid (18:1Δ⁶) in transgenic crop plants. Thisfatty acid is useful in the production of low-caloric margarine and as aprecursor of adipic acid for the manufacture of nylon 66 (Murphy, (1992)Trends Biotechnol. 10, 84-87; and Ohlrogge, (1994) Plant Physiol. 104,821-826).

Also, the rationally designed L118F/P179I mutant of the castor Δ⁹-18:0-ACP desaturase may be useful in enhancing the unsaturated fattyacid quality of vegetable oils through the reduction of palmitic acid(16:0) content. These results along with additional information fromcrystallographic studies of acyl-ACP desaturases will lead to thecontinued design of enzymes for the production of novel monounsaturatedfatty acids in transgenic oilseed crops.

Material and Methods

Preparation of Chimeric Enzymes and Site-directed Mutants of the Δ⁶-16:0-ACP Desaturase

Chimeric enzymes were prepared by linking portions of the codingsequence of the mature T. alata Δ⁹ -18:0- and Δ⁶ -16:0-ACP desaturasesvia native restriction enzyme sites or restriction sites generated byPCR. The coding sequences used were derived from the previouslydescribed cDNAs pTAD2 (Cahoon et al., (1994) Plant Physiol. 106,807-808) and pTAD4 (Cahoon et al., (1994) J. Biol. Chem. 269,27519-27526). Site-specific mutations in the coding sequence of aminoacids 178-202 of the mature Δ⁹ -18:0-ACP desaturase (equivalent toresidues 172-196 of the Δ⁶ -16:0-ACP desaturase) were introduced byextension and amplification of overlapping oligonucleotide primers usingPCR with Pfu polymerase (Stratagene). As an example, mutationsA188G/Y189F were made with the following oligonucleotides (base changesare indicated in upper case): 5'atggatcctggcacggataacaacccgtac3' ---SEQID NO: 1--- (Primer 1); 5'acgaggtgtagataAatCcgaggtacgggttgttatccg3'---SEQ ID NO: 2--- (Primer 2); 5'tatctacacctcgtatcaggagagggcgaca3'---SEQ ID NO: 3--- (Primer 3); 5'ttgaattccatgggaaatcgctgtcgccctctcctg3'---SEQ ID NO: 4--- (Primer 4). PCR reactions were conducted withoutadded template using 12.5 pmoles of Primers 1 and 4 and 6.25 pmoles ofPrimers 2 and 3 in 50 μl reactions. For the first 10 PCR cycles, anannealing temperature of 37° C. and an extension temperature of 72° C.were used. This was followed by an additional 20 cycles with theannealing temperature increased to 55° C. Other mutations in the regionof amino acids 178-202 were generated using a similar combination ofprimers containing coding sequence of the wild type enzyme or that ofthe desired mutation. PCR products were linked at their 5' and 3' endsto the coding sequence of amino acids 1-171 and 197-355 of the wild typeΔ⁶ -16:0-ACP desaturase via 5' BamHI and 3' NcoI sites and assembledinto the bacterial expression vector pET3d (Novagen). The mutationS205N/L206T/G207A was generated by PCR amplification of the codingsequence of amino acids 197-355 of the Δ⁶ -16:0-ACP desaturase using astemplate a cDNA for this enzyme in pBluescript II SK(-) (Jaworski andStumpf (1974) Arch. Biochem. Biophys. 162, 158-165). The 5'oligonucleotide (5'rtttccatgggaACACggCTcggctagcgaggcagaagg3' ---SEQ IDNO: 8---) contained the appropriate mutant codons (shown in upper case),and the T7 primer was used as the 3' oligonucleotide for PCR reactions.The amplification product was digested with NcoI and BclI and insertedinto the NcoI/amHI site of pET3d. A Ncol/EcoRI fragment from thisconstruct was then ligated to the coding sequence of amino acids 1-196of wild type or mutant Δ⁶ -16:0 ACP desaturases (e.g., A181T/A200F) togenerate a full-length coding sequence. All PCR generated DNA wassequenced using a Sequenase 2.0 kit (Amersham) to confirm the presenceof desired mutations and the absence of any secondary mutations.

Preparation of Site-directed Mutants of the Castor Δ⁹ -18:0-ACPDesaturase

Mutations L118F and P179I were introduced sequentially into the castorΔ⁹ -18:0-ACP desaturase. The mutation L118F was generated by overlapextension PCR (Ho et al., (1989) Gene 77, 51-59) using the codingsequence of the mature wild type castor Δ⁹ -18:0-ACP desaturase in thevector pET9d (Novagen) as the template. Two separate PCR reactions wereconducted using the primer pairs (base changes are shown in upper case):(a) T7 primer and 5'ccgaactccatcGaaggtattcagca3' ---SEQ ID NO: 9--- and(b) 5'tgctgaataccttCgatggagttcgg3' ---SEQ ID NO: 10--- and5'gcaaaagccaaaacggtaccatcaggatca3' ---SEQ ID NO: 11---(Primer A). Theagarose gel-purified products of the two reactions were combined andamplified together with the T7 primer and Primer A. The product of thisreaction containing the mutation for L118F was digested with XbaI andBamHI and inserted in place of the corresponding portion of the wildtype castor Δ⁹ -18:0-ACP desaturase in the vector pET9d.

The double mutant L118F/P179T was generated as described above using thecoding sequence of the mutant L118F in pET9d as template and the primerpairs (base changes in upper case): (a) T7 primer and5'ggactgttttctgtccgAATatccattcctqaacca3' ---SEQ ID NO: 12---- and (b)5'tggttcaggaatggatATTcggacagaaaacagtcc3' ---SEQ ID NO: 13--- and PrimerA. The product generated following two rounds of PCP was digested withXbaI and PstT and inserted in place of the corresponding portion of thewild type castor Δ⁹ -18:0-ACP desaturase in vector pET9d.

Expression and Purification of Acyl-ACP Desaturases

Wild type and mutant acyl-ACP desaturases were obtained by expression ofthe coding sequences in Escherichia coli BL21 (DE3) behind the T7 RNApolymerase promoter using pET expression vectors (Novagen) as describedabove. Recombinant enzymes whose activities are described in Table 1were purified from extracts of six to nine liter bacterial culturesinduced at 22° C. (or at 30° C., in the case of the wild type castor Δ⁹-18:0-ACP desaturase and mutant L118F/P179I). Protein purification wasperformed using DEAE-anion exchange chromatography followed by perfusioncation exchange chromatography using a 20 HS column (PerSeptiveBiosystems). Mutant desaturases were obtained at 90% purity, and thewild-type Δ⁶ -16:0-ACP desaturase was recovered at approximately 80%purity as determined by SDS-PAGE with Coomassie staining. The wild-typecastor Δ⁹ -18:0-ACP desaturase and mutant L118F/P179T were enrichedto >90% purity using only 20 HS perfusion cation exchangechromatography. Following purification, enzymes were exchanged into abuffer consisting of 40 mM Tris-HCl (pH 7.5), 40 mM NaCl, and 10%glycerol and stored in aliquots at -75° C.

Enzyme Assays

Acyl-ACP desaturation assays and analysis of reaction products wereconducted as previously described (Cahoon et al., (T1994) J. Biol. Chem.269, 27519-27520) with the following modifications: recombinant Anabaenavegetative ferredoxin (22 μg/assay) and maize root ferredoxin-NADP⁺oxidoreductase (FNR) (0.4 U/assay) were used in place of spinachferredoxin and FNP, and concentrations of NADPH and [1-¹⁴ C]16:0- or18:0- ACP per assay were increased to 2.5 mM and 1.2 μM, respectively.Acyl-ACPs were synthesized enzymatically (using recombinant spinachACP-I. Products and unreacted substrates were separated by argentationthin layer chromatography (TLC) as methyl ester derivatives (Cahoon andOhlrogge, (1994) Plant Physiol. 104, 827-844; Cahoon et al., (1994) J.Biol. Chem. 269, 27519-27526; and Morris et al., (1967) J.Chromatography 31, 69-76), and the distribution of radioactivity betweenthese moieties was measured by phosphorimaging of TLC plates usingImageQuant software (Molecular Dynamics) and by liquid scintillationcounting of TLC scrapings.

Determination of Double Bond Positions

Double bond positions of monounsaturated fatty acid products weredetermined by the mobility of methyl ester derivatives on 15%argentation TLC plates (Cahoon and Ohlrogge, (1994) Plant Physiol. 104,827-844; and Morris et al., (1967) J. Chromatography 31, 69-76) and bygas chromatography-mass spectrometry (GC-MS) of dimethyl disulfideadducts of these derivatives (Cahoon et al., (1992) Proc. Natl. Acad.Sci. U.S.A. 89, 11184-11188; Cahoon et al., (1994) J. Biol. Chem. 269,27519-27526; and Yamamoto et al., (1991) Chem. Phys. Lipids 60, 39-50).Desaturation assays for GC-MS analyses were conducted usingradiolabelled 16:0- and 18:0-ACP and unlabelled 17:0-ACP as substratesfor purified enzymes.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 14                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 #           30     ATAA CAACCCGTAC                                            - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 #    39            TCCG AGGTACGGGT TGTTATCCG                                  - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #          31      CAGG AGAGGGCGAC A                                          - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #       36         ATCG CTGTCGCCCT CTCCTG                                     - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 #           30     ATAA CAACCCGTAC                                            - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #    39            TGCG AGGTACGGGT TGTTATCCG                                  - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #       36         ATGA ATGTCGCCCT CTCCTG                                     - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 38 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #     38           GCTC GGCTAGCGAG GCAGAAGG                                   - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 #              26  GTAT TCAGCA                                                - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                #              26  TGGA GTTCGG                                                - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #           30     TACC ATCAGGATCA                                            - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #       36         GAAT ATCCATTCCT GAACCA                                     - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #       36         ATTC GGACAGAAAA CAGTCC                                     - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 30 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - Asp Pro Gly Ala Asp Asn Asn Pro Tyr Leu Al - #a Tyr Ile Tyr Thr Ser         #                15                                                           - Tyr Gln Glu Arg Ala Thr Ala Ile Ser His Gl - #y Ser Leu Gly                 #            30                                                               __________________________________________________________________________

We claim:
 1. A method for modifying the chain length and double bondpositional specificities of a soluble plant fatty acid desaturase, themethod comprising modifying one or more amino acid residues in thesubstrates binding channel of the soluble plant fatty acid desaturasewhich do not make direct contact with substrate.
 2. A method formodifying the chainlength and double bond specificities of a solubleplant fatty acid desaturase, the method comprising modifying the aminoacid residue corresponding to amino acid 200 of the Ricinus communis Δ⁹ACP desaturase.
 3. The method of claim 1 wherein the amino acid residuesare located at the upper part of the substrate binding channel of thesoluble fatty acid desaturase.
 4. The method of claim 3 wherein thesoluble plant fatty acid desaturase is an acyl-ACP desaturase.
 5. Themethod of claim 4 wherein the acyl-ACP desaturase is a Δ⁹ desaturase. 6.The method of claim 5 wherein the Δ⁹ desaturase is produced by a plantselected from the group consisting of Thunbergia alata or Ricinuscommunis.
 7. The method of claim 6 wherein the amino acid residues areselected from the group consisting of residues corresponding to aminoacids 203, 204, 205, 206 and 207 of the Ricinus communis Δ⁹ ACPdesaturase.
 8. A method for modifying the chain length and double bondpositional specificities of an acyl-ACP desaturase, comprising:a)providing the primary amino acid sequence of the acyl-ACP desaturase; b)aligning the primary amino acid sequence of the acyl-ACP desaturase withthe primary amino acid sequence of the Ricinus communes Δ⁹ desaturasefor maximum sequence conservation; c) constructing a 3-dimensional modelfor the acyl-ACP desaturase based on the sequence conservation with theRicinus communis Δ⁹ desaturase; d) identifying amino acid residues whichmost closely correspond to amino acids 200, 203, 204, 205, 206, and 207of the Ricinus communis Δ⁹ desaturase, of the structure modeled in stepc); and e) generating a mutant acyl-ACP desaturase having modified chainlength and double bond positional specificities by replacing one or moreof the amino acid residues identified in step d) with another amino acidresidue.
 9. A mutant acyl-ACP desaturase which is characterized by theability to catalyze desaturation of a first fatty acid and a secondfatty acid, the first and second fatty acids differing in their chainlength, the desaturation rates of both the first and second fatty acidsdiffering by no more than about 4-fold, the mutant containing a pointmutation at one or more amino acid residues which do not make directcontact with substrate.
 10. The mutant acyl-ACP desaturase of claim 9wherein the first fatty acid has a chain-length of 16:0 and the secondfatty acid has a chain-length of 18:0.
 11. The mutant acyl-ACPdesaturase of claim 9 wherein the amino acid residues which do not makedirect contact with substrate are selected from the group consisting ofresidues corresponding to amino acids 200, 203, 204, 205, 206 and 207 ofthe Ricinus communis Δ⁹ desaturase.
 12. A mutant acyl-ACP desaturasehaving one or more amino acid substitutions at residues which do notmake direct contact with substrate, which is characterized by changes inchain-length and double bond positional specificity as compared to thewild-type acyl-ACP desaturase counterpart.
 13. The mutant acyl-ACPdesaturase of claim 12 wherein the acyl-ACP desaturase is the Δ⁹acyl-ACP desaturase and the residue corresponds to amino acid 200 of theRicinus communis Δ⁹ desaturase.
 14. The mutant acyl-ACP desaturase ofclaim 12 wherein the residues are located at the upper part of thesubstrate binding channel.
 15. The mutant acyl-ACP desaturase of claim14 wherein the acyl-ACP desaturase is the Δ⁹ acyl-ACP desaturase and theresidues are selected from the group consisting of residuescorresponding to amino acids 203, 204, 205, 206 and 207 of the Ricinuscommunis Δ² ACP desaturase.
 16. The mutant of claim 13 or claim 15wherein the Δ⁹ acyl-ACP desaturase is produced by mutagenizing nucleicacid cloned from Thunbergia alata or Ricinus communis.
 17. A nucleicacid sequence encoding a mutant acyl-ACP desaturase which ischaracterized by the ability to catalyze desaturation of a first fattyacid and a second fatty acid, the first and second fatty acids differingin their chain length, the desaturation rates of both the first andsecond fatty acids differing by no more than about 4-fold, the nucleicacid sequence encoding the mutant acyl-ACP desaturase beingcharacterized by a point mutation at one or more amino acid residueswhich do not make direct contact with substrate, the nucleic acidsequence being further characterized as having a sufficient degree ofamino acid identity with the amino acid sequence of Ricinus communis Δ⁹desaturase to enable statistically significant sequence alignment withthe Ricinus communis Δ⁹ desaturase.
 18. The nucleic acid sequence ofclaim 17 wherein the point mutation is introduced into wild-type Ricinuscommunis Δ⁹ desaturase at the residue corresponding to reside 200 of theRicinus communis Δ⁹ ACP desaturase.
 19. The nucleic acid sequence ofclaim 17 wherein the amino acid residue is located at the upper part ofthe substrate binding channel of the acyl-ACP desaturase.
 20. Thenucleic acid sequence of claim 19 wherein the point mutation isintroduced into wild-type Ricinus communis Δ⁹ desaturase at one or moreamino acid residues selected from the group consisting of residuescorresponding to amino acids 203, 204, 205, 206 and 207 of the Ricinuscommunis Δ⁹ ACP desaturase.
 21. A DNA expression construct comprising,in expressible form, a nucleic acid sequence encoding a mutant acyl-ACPdesaturase which is characterized by the ability to catalyzedesaturation of a first fatty acid and a second fatty acid, the firstand second fatty acids differing in their chain length, the desaturationrates of both the first and second fatty acids differing by no more thanabout 4-fold, the nucleic acid sequence encoding the mutant acyl-ACPdesaturase being characterized by a point mutation at one or more aminoacid residues which do not make direct contact with substrate, thenucleic acid sequence being further characterized as having a sufficientdegree of amino acid identity with the amino acid sequence of Ricinuscommunis Δ⁹ desaturase to enable statistically significant sequencealignment with the Ricinus communis Δ⁹ desaturase.
 22. The DNAexpression construct of claim 21 wherein the point mutation isintroduced into wild-type Ricinus communis Δ⁹ desaturase at the residuecorresponding to residue 200 of the Ricinus communis Δ⁹ ACP desaturase.23. The DNA expression construct of claim 21 wherein the amino acidresidue is located at the upper part of the substrate binding channel ofthe acyl-ACP desaturase.
 24. The DNA expression construct of claim 23wherein the point mutation is introduced into wild-type Ricinus communisΔ⁹ desaturase at one or more amino acid residues selected from the groupconsisting of residues corresponding to residue 203, 204, 205, 206 and207 of the Ricinus communis Δ⁹ ACP desaturase.
 25. A cell transformedwith a DNA expression construct comprising, in expressible form, anucleic acid sequence encoding a mutant acyl-ACP desaturase which ischaracterized by the ability to catalyze desaturation of a first fattyacid and a second fatty acid, the first and second fatty acids differingin their chain length, the desaturation rates of both the first andsecond fatty acids differing by no more than about 4-fold, the nucleicacid sequence encoding the mutant acyl-ACP desaturase beingcharacterized by a point mutation at one or more amino acid residueswhich do not make direct contact with substrate, the nucleic acidsequence being further characterized as having a sufficient degree ofamino acid identity with the amino acid sequence of Ricinus communis Δ⁹desaturase to enable statistically significant sequence alignment withthe Ricinus communis Δ⁹ desaturase.
 26. The cell of claim 25 wherein thepoint mutation is introduced into wild-type Ricinus communis Δ⁹desaturase at the amino acid residue corresponding to residue 200 of theRicinus communis Δ⁹ ACP desaturase.
 27. The cell of claim 25 wherein theamino acid residue is located at the upper part of the substrate bindingchannel of the acyl-ACP desaturase.
 28. The cell of claim 27 wherein thepoint mutation is introduced into wild-type Ricinus communis Δ⁹desaturase at one or more amino acid residues selected from the groupconsisting of residues corresponding to residue 203, 204, 205, 206 and207 of the Ricinus communis Δ⁹ ACP desaturase.
 29. The cell of claim 25which is a prokaryotic cell.
 30. The cell of claim 25 which is aeukaryotic cell.
 31. The cell of claim 30 which is a plant cell.
 32. ADNA expression construct comprising, in expressible form, a nucleic acidsequence encoding a chimeric acyl-ACP desaturase which is characterizedby the ability to catalyze desaturation of a first fatty acid and asecond fatty acid, the first and second fatty acids differing in theirchain length, the desaturation rates of both the first and second fattyacids differing by no more than about 4-fold.
 33. The DNA expressionconstruct of claim 32 wherein the chimeric acyl-ACP desaturase comprisesΔ⁶ -16:0 in which amino acids corresponding to amino acids 172-201 ofThunbergia alata Δ⁶ -16:0 ACP desaturase are replaced with amino acidscorresponding to amino acids 178-207 of the Thunbergia alata Δ⁹ -18:0ACP desaturase from a Δ⁹ -18:0 ACP desaturase.
 34. The DNA expressionconstruct of claim 32 wherein the chimeric acyl-ACP desaturase comprisesΔ⁶ -16:0 in which amino acids corresponding to amino acids 172-196 ofThunbergia alata Δ⁶ -16:0 ACP desaturase are replaced with amino acidscorresponding to amino acids 178-202 of the Thunbergia alata Δ⁹ -18:0ACP desaturase from a Δ⁹ -18:0 ACP desaturase.
 35. The DNA expressionconstruct of claim 32 wherein the chimeric acyl-ACP desaturase comprisesa Δ⁶ -16:0 ACP desaturase in which amino acids corresponding to aminoacids 176, 183, 184, 200, 201 and 202 of the Thunbergia alata Δ⁶ -16:0ACP desaturase are replaced with amino acids corresponding to 181, 188,189, 205, 206 and 207 of the Thunbergia alata Δ⁹ -18:0 ACP desaturase,respectively, from a Δ⁹ -18:0 ACP desaturase.
 36. The DNA expressionconstruct of claim 32 wherein the chimeric acyl-ACP desaturase comprisesa Δ⁶ -16:0 ACP desaturase in which amino acids 183 and 184 of theThunbergia alata Δ⁶ -16:0 ACP desaturase are replaced with amino acidscorresponding to amino acids 188 and 189 of the Thunbergia alata Δ⁹-18:0 ACP desaturase, respectively, from a Δ⁹ -18:0 ACP desaturase. 37.The DNA expression construct of claim 35 wherein the chimeric acyl-ACPdesaturase comprises a Δ⁶ -16:0 ACP desaturase in which amino acidscorresponding to amino acids 176 and 195 of the Thunbergia alata Δ⁶-16:0 ACP desaturase are replaced with amino acids corresponding to 181and 200 of the Thunbergia alata Δ⁹ -18:0 ACP desaturase, respectively,from a Δ⁹ -18:0 ACP desaturase.
 38. The DNA expression construct ofclaim 35 wherein the chimeric acyl-ACP desaturase comprises a Δ⁶ -16:0ACP desaturase in which amino acids corresponding to amino acids 176,195, 200, 201 and 202 of the Thunbergia alata Δ⁶ -16:0 ACP desaturaseare replaced with amino acids corresponding to amino acids 181, 200,205, 206 and 207 of the Thunbergia alata Δ⁹ -18:0 ACP desaturase,respectively, from a Δ⁹ -18:0 ACP desaturase.
 39. A method for modifyingthe chain length and double bond positional specificities of a solubleplant fatty acid desaturase, the method comprising modifying one or moreamino acid contact residues in the substrate binding channel of thesoluble fatty acid desaturase which contact the fatty acid, andmodifying one or more amino acids which do not contact substrate. 40.The method of claim 39 wherein the soluble plant fatty acid desaturaseis an acyl-ACP desaturase.
 41. The method of claim 40 wherein theacyl-ACP desaturase is a Δ⁹ desaturase.
 42. The method of claim 41wherein the Δ⁹ desaturase is produced by a plant selected from the groupconsisting of Thunbergia alata or Ricinus communis.
 43. The method ofclaim 42 wherein the amino acid contact residues are selected from thegroup consisting of residues corresponding to amino acids 114, 115, 117,118, 179, 181, 188 and 189 of the Ricinus communis Δ⁹ ACP desaturase andthe amino acids which do not contact substrate are selected from thegroup consisting of residues corresponding to amino acids 200, 203, 204,205, 206, and 207 of the Ricinus communis Δ⁹ ACP desaturase.
 44. Amethod for modifying the chain length and double bond positionalspecificities of an acyl-ACP desaturase, comprising:a) providing theprimary amino acid sequence of the acyl-ACP desaturase; b) aligning theprimary amino acid sequence of the acyl-ACP desaturase with the primaryamino acid sequence of the Ricinus communis Δ⁹ desaturase for maximumsequence conservation; c) constructing a 3-dimensional model for theacyl-ACP desaturase based on the sequence conservation with the Ricinuscommunis Δ⁹ desaturase; d) identifying amino acid contact residueswithin the substrate binding channel of the structure modeled in stepc); e) identifying amino acid residues which most closely correspond tothe amino acids 200, 203, 204, 205, 206, and 207 of the Ricinus communisΔ⁹ ACP desaturase of the structure modeled in step c); and f) generatinga mutant acyl-ACP desaturase having modified chain length and doublebond positional specificities by replacing one or more of the amino acidcontact residues identified in step d) with another amino acid residue,and replacing one or more of the amino acid residues identified in stepe) with another amino acid residue.
 45. A mutant acyl-ACP desaturasewhich is characterized by the ability to catalyze desaturation of afirst fatty acid and a second fatty acid, the first and second fattyacids differing in their chain length, the desaturation rates of boththe first and second fatty acids differing by no more than about 4-foldwhich contains a point mutation at one or more amino acid contactresidues in the substrate binding channel and also contains a pointmutation at one or more amino acid residues which do not make directcontact with substrate.
 46. The mutant acyl-ACP desaturase of claim 45wherein the first fatty acid has a chain-length of 16:0 and the secondfatty acid has a chain-length of 18:0.
 47. A mutant acyl-ACP desaturasehaving one or more amino acid substitutions at contact residues withinthe substrate binding channel and one or more amino acid substations atresidues which does not make direct contact with substrate.
 48. Themutant acyl-ACP desaturase of claim 47 which is characterized by changesin chain-length and double bond positional specificity as compared tothe wild-type acyl-ACP desaturase counterpart.
 49. The mutant acyl-ACPdesaturase of claim 48 wherein the acyl-ACP desaturase is the Δ⁹acyl-ACP desaturase and the contact residues within the substratebinding channel are selected from the group consisting of residuescorresponding to amino acids 114, 115, 117, 118, 179, 181, 188 and 189of the Ricinus communis Δ⁹ ACP desaturase, and the residue which doesnot make direct contact with substrate is selected from the groupconsisting of residues corresponding to amino acids 200, 203, 204, 205,206 and 207 of the Ricinus communis Δ⁹ ACP desaturase.
 50. The mutant ofclaim 49 wherein the Δ⁹ acyl-ACP desaturase is produced by mutagenizingnucleic acid cloned from Thunbergia alata or Ricinus conmmunis.
 51. Anucleic acid sequence encoding a mutant acyl-ACP desaturasecharacterized by the ability to catalyze desaturation of a first fattyacid and a second fatty acid, the first and second fatty acids differingin their chain length, the desaturation rates of both the first andsecond fatty acids differing by no more than about 4-fold which containsa point mutation at one or more amino acid contact residues in thesubstrate binding channel and further contains a point mutation at oneor more amino acid residues which do not make direct contact withsubstrate, the nucleic acid sequence being further characterized ashaving a sufficient degree of amino acid identity with the amino acidsequence of Ricinus communis Δ⁹ desaturase to enable statisticallysignificant sequence alignment with the Ricinus communis Δ⁹ desaturase.52. The nucleic acid sequence of claim 51 wherein the first fatty acidhas a chain-length of 16:0 and the second fatty acid has a chain-lengthof 18:0.
 53. The nucleic acid sequence of claim 52 wherein the acyl-ACPdesaturase is the Δ⁹ acyl-ACP desaturase and the contact residues withinthe substrate binding channel are selected from the group consisting ofresidues corresponding to amino acids 114, 115, 117, 118, 179, 181, 188and 189 of the Ricinus communis Δ⁹ ACP desaturase, and the residue whichdoes not make direct contact with substrate is selected from the groupconsisting of residues corresponding to amino acids 200, 203, 204, 205,206 and 207 of the Ricinus communis Δ⁹ ACP desaturase.
 54. A celltransformed with a DNA expression construct comprising, in expressibleform, a nucleic acid sequence encoding a mutant acyl-ACP desaturasewhich is characterized by the ability to catalyze desaturation of afirst fatty acid and a second fatty acid, the first and second fattyacids differing in their chain length, the desaturation rates of boththe first and second fatty acids differing by no more than about 4-fold,the nucleic acid sequence encoding the mutant acyl-ACP desaturase beingcharacterized by a point mutation at one or more amino acid contactresidues in the substrate binding channel, and one or more amino acidresidues which do not make direct contact with substrate, the nucleicacid sequence being further characterized as having a sufficient degreeof amino acid identity with the amino acid sequence of Ricinus communisΔ⁹ desaturase to enable statistically significant sequence alignmentwith the Ricinus communis Δ⁹ desaturase.
 55. The cell of claim 54wherein the amino acid contact residues are selected from the groupconsisting of residues corresponding to amino acids 114, 115, 117, 118,179, 181, 188 and 189 of the Ricinus communis Δ⁹ ACP desaturase and theamino acid residues which do not make direct contact with substrate areselected from the group consisting of residues corresponding to aminoacids 200, 203, 204, 205, 206 and 207 of the Ricinus communis Δ⁹ ACPdesaturase.
 56. The cell of claim 54 which is a prokaryotic cell. 57.The cell of claim 54 which is a eukaryotic cell.
 58. The cell of claim57 which is a plant cell.